Evidence for the presence of diacylglycerol kinase in rat brain myelin

The previous demonstration that incubation of brain slices with [³²P]phosphate brings about rapid tabeling of phosphatidic acid in myelin suggests that the enzyme involved should be present in this specialized membrane. DAG kinase (ATP:1,2-diacyglycerol 3-phosphotransferase, E.C. 2.7.1.107) is present in rat brain homogenate at a specific activity of 2.5 nmol phosphatidic acid formed/min/mg protein, while highly purified myelin had a much lower specific activity (0.29 nmol/min/mg protein). Nevertheless, the enzyme appears to be intrinsic to this membrane since it can not be removed by washing with a variety of detergents or chelating agents, and it could not be accounted for as contamination by another subcellular fraction. Production of endogenous, membrane-associated, diacylglycerol (DAG) by PLC (phospholipase C) treatment brought about translocation from soluble to particulate fractions, including myelin. Another level of control of activity involves inactivation by phosphorylation; a 10 min incubation of brain homogenate with ATP resulted in a large decrease in DAG kinase activity in soluble, particulate and myelin fractions.     

Protein Phosphorylation Induced by Phorbol Esters and Cyclic AMP in Anterior Pituitary Cells: Possible Role in Adrenocorticotropin Release and Synthesis

Forskolin, an activator of adenylate cyclase, stimulates adrenocorticotropin (ACTH) release and increases proopiomelanocortin mRNA levels in anterior pituitary cells by enhancing cyclic AMP (cAMP)-dependent protein kinase activity. The phorbol ester phorbol 12-myristate 13-acetate (PMA) evokes these same responses from anterior pituitary cells by activating protein kinase C. Both protein kinases most likely induce their cellular effects by catalyzing the phosphorylation of specific proteins. To elucidate the mechanisms by which cAMP-dependent protein kinase and protein kinase C promote ACTH secretion and synthesis, the phosphoproteins regulated by forskolin and PMA were identified in the cell line AtT-20, which consists of a homogeneous population of corticotrophs. Phosphoproteins were analyzed in different subcellular fractions by two-dimensional polyacrylamide gel electrophoresis and autoradiography. Forskolin increased phosphate incorporation into two proteins in the cytoplasmic fraction of 24 kilodaltons (kd) (pI 6.8) and 40 kd (pI 5.8), two proteins in the plasma membrane fraction of 32 kd (pI 8.3) and 60 kd (pI 8), and one protein in the nuclear fraction of 20 kd (pI 8.7). Insertion of the inhibitor of cAMP-dependent protein kinase into the AtT-20 cells, using a liposome technique, blocked the rise in phosphate incorporation induced by forskolin. PMA also stimulated phosphate incorporation into proteins in AtT-20 cells. PMA increased the phosphorylation of three cytoplasmic proteins of 25 kd (pI 7.6), 40 kd (pI 5.8), and 40 kd (pI 8.1) as well as two membrane proteins of 32 kd (pI 8.3) and 60 kd (pI 8) and one nuclear protein of 20 kd (pI 6.3).(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in ribulosebisphosphate carboxylase/oxygenase and ribulose 5-phosphate kinase abundances and photosynthetic capacity during leaf senescence

The abundances of ribulose-1,5-bisphosphate carboxylate/oxygenase (Rubisco) and ribulose-5-phosphate (Ru5P) kinase in field-grown soybean (Glycine max L. Merr.) leaves were quantified by a Western blot technique and related to changes in chlorophyll and photosynthetic capacity during senescence. Even though the leaf content of Rubisco was approximately 80-fold greater than that of Ru5P kinase, the decline in the levels of these two Calvin cycle enzymes occurred in parallel during the senescence of the leaves. Moreover, the decrease in the content of Rubisco was accompanied by parallel decreases of both the large and small subunits of this enzyme but not by an accumulation of altered large or small subunit isoforms. With increasing senescence, decreases in abundances of Rubisco, Ru5P kinase and chlorophyll were closely correlated with the decline in photosynthetic capacity; thus, the specific photosynthetic capacity when expressed per abundance of any of these parameters was rather constant despite an 8-fold decrease in photosynthetic capacity. These results suggest that during senescence of soybean leaves the chloroplast is subject to autolysis by mechanisms causing an approximately 80-fold greater rate of loss of Rubisco than Ru5P kinase.Jointly supported by the United States Department of Agricultural Research Service and the Kentucky Agricultural Experiment Station, Lexington (paper No. 88 3 286).Mention of a commercial product does not constitute endorsement by the United States Department of Agriculture.     

正常人及肝细胞癌患者肝脏及血浆中丙酮酸激酶同工酶免疫测定

 分别从人肝及鼠肝中高度纯化L型和M_1型丙酮酸激酶(Pyk),制备相应的免抗血清。从抗血清中纯化IgG并水解成F(ab')_2,进而还原成Fab',后者与辣根过氧物酶(HRP)交联成Fab'-HRP,再利用这两种复合物建立了各自的高灵敏夹心ELISA来免疫定量L及M_2型(与鼠M_1-Pyk有交叉免疫)Pyk,结果发现:正常人肝中95％的Pyk为L型,M_2-Pyk仅占5％,而肝细胞癌中L-Pyk降至正常肝的3.5％,M_2-Pyk却增至正常的14.6倍,以致M_2-Pyk占总量的95.5％。免疫组化研究进一步证实定量的结果,正常人肝L-Pyk染色呈强阳性。M_2-Pyk染色较弱,而肝细胞癌恰好相反,L-Pyk染色明显减退,而M_2-Pyk不仅癌组织染色很深,癌周组织也明显深于正常,而且愈近癌巢,染色愈深。测定血浆Pyk,发现正常人L-Pyk占89.9％,M-Pyk(以M_2计)仅占10.1％。肝细胞癌患者血浆L-Pyk略见降低,为正常值的79.6％,p值在0.02～0.05之间,但血浆M型Pyk明显升高至正常的4.59倍,占Pyk总量的39.6％,并证明主要来源于肝癌组织中的M_2,故M_2-Pyk有可能成为肝细胞癌诊断的新指标。     

Rat RI beta isoform of type I regulatory subunit of cyclic adenosine monophosphate-dependent protein kinase: cDNA sequence analysis, mRNA tissue specificity, and rat/mouse difference in expression in testis

A rat complementary DNA (cDNA) for the RI beta isoform of type I cyclic adenosine monophosphate (cAMP)-dependent protein kinase regulatory subunit was cloned and sequenced and was found to contain the entire protein coding and 3'-untranslated regions, with a single (ATTAAA) poly-adenylation site. The largest open reading frame was preceded by a short out-of-phase open reading frame, which is not seen in the corresponding mouse RI beta cDNA due to a single base substitution. The rat RI beta cDNA clone was 2,374 bases long and detected a rat mRNA of approximately 2.8 kilobases. Rat RI beta mRNA was abundant in adult rat brain and testis but was undetectable in other rat tissues. The rat RI beta cDNA also detected RI beta mRNA in mouse brain, but not mouse testis, from 10-week-old BALB/c or 10- and 6-week-old Swiss Webster mice. Thus, despite a 96% nucleotide identity in the coding region of RI beta in rat vs. mouse, there are at least two differences in these closely related species. First, there is a short open reading frame, which precedes the coding region in the rat but not the mouse. Second, unlike the mouse testis, the rat testis contains abundant levels of RI beta mRNA.     

Regulation of Tight Junction Resistance in T84 Monolayers by Elevation in Intracellular Ca2+: A Protein Kinase C Effect

Elevation in intracellular Ca²⁺ acting via protein kinase C (PKC) is shown to regulate tight junction resistance in T₈₄ cells, a human colon cancer line and a model Cl⁻ secretory epithelial cell. The Ca²⁺ ionophore A23187, which was used to increase the intracellular Ca²⁺ concentration, caused a decrease in tight junction resistance in a concentration- and time-dependent manner. Dual Na⁺/mannitol serosal-to-mucosal flux analysis performed across the T₈₄ monolayers treated with 2 μm A23187 revealed that A23187 increased both fluxes and that in the presence of ionophore there was a linear relationship between the Na⁺ and mannitol fluxes with a slope of 56.4, indicating that the decrease in transepithelial resistance was due to a decrease in tight junction resistance. Whereas there was no effect of 0.1 μm A23187, 1 or 2 μm produced a 55% decrease in baseline resistance in 1 hr and 10 μm decreased resistance more than 80%. The A23187-induced decrease in tight junction resistance was partially reversible by washing 3 times with a Ringer's-HCO₃ solution containing 1% BSA. The A23187 effect on resistance was dependent on intracellular Ca²⁺; loading the T₈₄ cells with the intracellular Ca²⁺ chelator BAPTA significantly reduced the decrease in tight junction resistance caused by A23187. This intracellular Ca²⁺ effect was mediated by protein kinase C and not calmodulin. While the protein kinase C antagonist H-7 totally prevented the action of A23187 on tight junction resistance, the Ca²⁺/calmodulin inhibitor W13 did not have any effect. Sphingosine, another inhibitor of PKC, partially reduced the A23187-induced decline in tight junction resistance. The PKC agonist PMA mimicked the A23187 effect on resistance, although the effect was delayed up to 1 hr after exposure. In addition, however, PMA also caused an earlier increase in resistance, indicating it had an additional effect in addition to mimicking the effect of elevating Ca²⁺. The effects of a phospholipase inhibitor (mepacrine) and of inhibitors of arachidonic acid metabolism (indomethacin for the cyclooxygenase pathway, NDGA for the lipoxygenase pathway, and SKF 525A for the epoxygenase pathway) on the A23187 action were also examined. None of these agents altered the A23187-induced decrease in resistance. Monolayers exposed to 2 μm A23187 for 1 hr were stained with fluorescein conjugated phalloidin, revealing that neighboring cells did not part one from another and that A23187 did not have a detectable effect on distribution of F-actin in the perijunctional actomyosin ring. The results indicate that elevation in intracellular Ca²⁺ decreases tight junction resistance in the T₈₄ monolayer, acting through protein kinase C by a mechanism which does not involve visible changes in the perijunctional actomyosin ring. Received: 14 July 1995/Revised: 25 September 1995     

Regulation of Ca2+-activated K+ Channel from Rabbit Distal Colon Epithelium by Phosphorylation and Dephosphorylation

The Ca²⁺-activated maxi K⁺ channel is predominant in the basolateral membrane of the surface cells in the distal colon. It may play a role in the regulation of the aldosterone-stimulated Na⁺ reabsorption from the intestinal lumen. Previous measurements of these basolateral K⁺ channels in planar lipid bilayers and in plasma membrane vesicles have shown a very high sensitivity to Ca²⁺ with a K _0.5 ranging from 20 nm to 300 nm, whereas other studies have a much lower sensitivity to Ca²⁺. To investigate whether this difference could be due to modulation by second messenger systems, the effect of phosphorylation and dephosphorylation was examined. After addition of phosphatase, the K⁺ channels lost their high sensitivity to Ca²⁺, yet they could still be activated by high concentrations of Ca²⁺ (10 μm). Furthermore, the high sensitivity to Ca²⁺ could be restored after phosphorylation catalyzed by a cAMP dependent protein kinase. There was no effect of addition of protein kinase C. In agreement with the involvement of enzymatic processes, lag periods of 30–120 sec for dephosphorylation and of 10–280 sec for phosphorylation were observed. The phosphorylation state of the channel did not influence the single channel conductance. The results demonstrate that the high sensitivity to Ca²⁺ of the maxi K⁺ channel from rabbit distal colon is a property of the phosphorylated form of the channel protein, and that the difference in Ca²⁺ sensitivity between the dephosphorylated and phosphorylated forms of the channel protein is more than one order of magnitude. The variety in Ca²⁺ sensitivities for maxi K⁺ channels from tissue to tissue and from different studies on the same tissue could be due to modification by second messenger systems. Received: 28 February 1995/Revised: 22 December 1995     

Evidence for Involvement of a Zymogen Granule Na+/H+ Exchanger in Enzyme Secretion from Rat Pancreatic Acinar Cells

We have characterized a Na⁺/H⁺ exchanger in the membrane of isolated zymogen granules (ZG) from rat exocrine pancreas and investigated its role in secretagogue-induced enzyme secretion. ZG Na⁺/H⁺ exchanger activity was estimated by measuring Na⁺ or Li⁺ influx and consequent osmotic swelling and lysis of ZG incubated in Na- or Li-acetate. Alternatively, intragranule pH was investigated by measuring absorbance changes in ZG which had been preloaded with the weak base acridine orange. Na⁺- or Li⁺-dependent ZG lysis was enhanced by increasing inward to outward directed H⁺ gradients. Na⁺-dependent ZG lysis was not prevented by an inside-positive K⁺ diffusion potential generated by valinomycin which argues against parallel operation of separate electrogenic Na⁺ and H⁺ permeabilities and for coupled Na⁺/H⁺ exchange through an electroneutral carrier. Na⁺- and Li⁺-dependent ZG lysis was inhibited by EIPA (EC₅₀∼25 μm) and benzamil (EC₅₀∼100 μm), but only weakly by amiloride. Similarly, absorbance changes due to release of acridine orange from acidic granules into the medium were obtained with Na⁺ and Li⁺ salts only, and were inhibited by EIPA, suggesting the presence of a Na⁺/H⁺ exchanger in the membrane. Na⁺ dependent lysis of ZG was inhibited by 0.5 mm MgATP and MgATP-γ-S by about 60% and 35%, respectively. Inhibition by MgATP was prevented by incubation of ZG with alkaline phosphatase (100 U/ml), or by the calmodulin antagonists calmidazolium (0.75 μm), trifluoperazine (100 μm) and W-7 (500 μm), suggesting that the ZG Na⁺/H⁺ exchanger is regulated by a ZG membrane-bound calmodulin-dependent protein kinase. Na⁺ dependence of secretagogue (CCK-OP)-stimulated amylase secretion was investigated in digitonin permeabilized rat pancreatic acini and was higher in acini incubated in Na⁺ containing buffer (30 mm NaCl/105 mm KCl buffer; 6.4 ± 0.4% of total amylase above basal) compared to buffer without Na⁺ (0 mm NaCl/135 mm KCl buffer; 4.7 ± 0.4% of total amylase above basal, P < 0.03). EIPA (50 μm) reduced CCK-OP-induced amylase secretion in Na⁺ containing buffer from 7.5 ± 0.6% to 4.1 ± 0.8% (P < 0.02). In the absence of Na⁺ in the buffer, CCK-OP-stimulated amylase release was not inhibited by 50 μm EIPA. The data suggest that an amiloride insensitive, EIPA inhibitable Na⁺/H⁺ exchanger is present in ZG membranes, which is stimulated by calmodulin antagonists and could be involved in secretagogue-induced enzyme secretion from rat pancreatic acini. Received: 7 December 1995/Revised: 2 April 1996